Coenzyme immobilized electrode

ABSTRACT

Improvements in a coenzyme electrode used to electrochemically measure the activity of enzyme or substrate concentration of the enzyme easily. The coenzyme is immobilized, without using a semipermeable membrane, on the electron collector directly with the chemical bond, particularly the covalent bond, whereby the activity of the oxido-reductase requiring the coenzyme can be measured. In addition, the oxido-reductase requiring the coenzyme is also immobilized together with such immobilized coenzyme to improve the characteristics of the conventional enzyme-coenzyme immobilized electrode.

This is a continuation of application Ser. No. 32,929, filed Apr. 19,1979 and now abandoned.

The present invention relates to improvements in coenzyme electrodes tobe used to electrochemically and easily measure the substrateconcentration of the enzyme. Also, the present invention relates toelectrodes for quickly measuring the activity of the enzyme.

The conventional example of the enzyme electrode composed of immobilizedoxidoreductase and coenzyme is described in literature (Anal. Chem.48(8), 1240, 1976). This is a sensor for lactic acid using lactatedehydrogenase (LDH) as the oxidoreductase, nicotineamide adeninedinucleotide (NAD) as the coenzyme, and carbon as electron collector. Asshown in FIG. 1, according to this principle, the lactic acid (AH2) asthe substrate is dehydrogenated (oxidized) by the enzyme catalyticreaction, and a hydrogen atom (electron) moves to NAD (oxidized) toproduce anodic current when the resultant NADH (reduced NAD) is directlyoxidized on the electron collector. Since the oxidation current value ofthe NADH varies in accordance with the concentration of the lactic acidas the substrate, the substrate concentration can be obtained from themeasurement of the anodic current.

In the above-described literature, the following two types ofconstructions are shown as the electrode with the enzyme and coenzymebeing immobilized.

(1) As shown in FIG. 2, an electrode wherein the LDH 1 and NAD 2 arecrosslinked on semipermeable membrane 5 with glutaraldehyde, and theenzyme and coenzyme immobilized on the surface of the membrane arephysically brought into contact against the carbon electron collector 3.

(2) As shown in FIG. 3, an electrode wherein agarose, which is one typeof polysaccharide, is used as polymer carrier 6, the NAD is immobilizedon the carrier, and the macro-molecular NAD is adapted to be retained,together with the enzyme, in the space between the carbon electroncollector 3 and the semipermeable membrane covering the carbon electroncollector. In this case, the enzyme is not chemically modifiedparticularly. In FIG. 3, reference numberal 4 represents a covalentbond.

Both of the above described enzyme electrodes (1) and (2) have thefollowing problems.

(a) In the case of measuring the activity of an enzyme, the enzymeitself cannot be diffused into the semipermeable membrane because thesurface of the electron collector is covered with the semipermeablemembrane. Thus, the activity of the enzyme cannot be measured by theelectrode with only the coenzyme, except for the enzyme, beingimmobilized.

(b) In the case of measuring the lactic acid concentration, it takestime for the lactic acid as the substrate to pass the semipermeablemembrane for diffusion thereinto and to come into contact against theenzyme. Thus, it takes time from injection of a lactic acid containingliquid as a specimen to be measured, to provision of the measured value(more than ten minutes), and the response current value to be obtainedis small.

(c) The response current value to be obtained is small. Accordingly, theanalytical sensitivity is lower.

The present invention is created to solve the problems of theabove-described enzyme electrode and, according to the presentinvention, there is provided a coenzyme immobilized electrode comprisinga coenzyme of oxidoreductase, and an electron collector, said coenzymebeing supported through covalent bonding on said electron collector oron a carrier which is mixed with said electron collector.

The coenzyme immobilized electrode of the present invention ischaracterized in that the coenzyme is immobilized, without using thesemipermeable membrane, on the electron collector directly, or on thecarrier which is mixed with the electron collector, whereby the activityof the oxidoreductase requiring the coenzyme can be measured. Inaddition, the oxidoreductase requiring the coenzyme is also immobilizedtogether with such immobilized coenzyme as described hereinabove toimprove the characteristics of the conventional enzyme-coenzymeimmobilized electrode.

These objects and advantages will be more readily apparent from thedetailed description in conjunction with the preferred embodiments andthe following drawings in which:

FIG. 1 is a typical view showing the relationship between enzymereaction and electrode reaction;

FIG. 2 and FIG. 3 are the typical views each showing the construction ofthe conventional enzyme-coenzyme immobilized electrode as referred toabove;

FIG. 4, FIG. 10, FIG. 11 and FIG. 12 are respectively the typical viewseach showing the construction of the coenzyme-enzyme immobilizedelectrode of the present invention;

FIG. 5 is a schematic view of a measuring system to be employed for theelectrode of the present invention;

FIG. 6 is a graph showing the anodic current response of the NADimmobilized electrode through addition of LDH in accordance with thepresent invention;

FIG. 7 shows the relationship between the LDH activity and the anodiccurrent increase;

FIG. 8 shows the anodic current of the NAD-LDH immobilized electrodethrough addition of lactic acid; and

FIG. 9 shows the relationship between the lactic acid concentration andanodic current increase.

The present invention will be described hereinafter with reference tothe embodiments.

EMBODIMENT 1

SnO₂ nesa-glass (semiconductive SnO₂ thin membrane attached on glassplate) is used as the electron collector, the LDH as the enzyme, and theNAD as the coenzyme.

As shown in the following equation, the NAD reacts with succinicanhydride in dimethyl sulfoxide (DMSO) and a carboxyl group isintroduced into the molecule. ##STR1##

The NAD derivative (I) reacts with the hydroxyl group on the SnO₂surface in the presence of dicyclohexyl carbodiimide (DCC) to produce anester bond, thereby to immobilize the NAD on the SnO₂ nesa-glasssurface. ##STR2##

The NAD immobilized SnO₂ nesa-glass electrode produced in theabove-described manner is dipped in a solution containing the LDH toproduce a complex of the NAD and the LDH. It is treated with across-linking reagent such as glutaraldehyde or the like to furtherimmobilize the LDH on the electron collector. In this case, the LDHforms the complex thereof with the NAD and are bonded, with each other,with the cross-linking reagent, and is immobilized on the electroncollector. The construction of the NAD-LDH immobilized electrodeproduced is shown in FIG. 4, wherein there are an enzyme 1 having asubstrate bonding site 1a and the coenzyme bonding site 1b, a coenzyme2, an electron collector 3 and covalent bond 4.

FIG. 5 shows a measuring system wherein the above-described coenzymeimmobilized electrode or the coenzyme-enzyme immobilized electrode areincorporated. Referring to FIG. 5, there are shown the above-describedtwo types of electrodes 7 with the lead wire 7' thereof, a saturatedcalomel electrode 8 for a reference electrode with the lead wire 8'thereof, a counter electrode 9 with the lead wire 9' thereof, a buffersolution 10, a separator 11, and an electrolytic cell 12.

A case where the above-described coenzyme electrode is used as theelectrode 7 to measure the enzyme activity will be describedhereinafter. The electrode 7 is set to a constant-potential of 0.4 Vwith respect to the electrode 8, using a potentionstat. Add the LDH intothe buffer solution containing 0.1 mol per l lactate, and the anodiccurrent flowing between the electrode 7 and the counter-electrode 9increases.

FIG. 6 shows the anodic current response when the LDH has been addedinto the buffer solution so that the LDH activity in the buffer solutionmay become 1 U/ml. The current shows a steady state value inapproximately two minutes after the addition of the LDH, and indicatesthe current increase of 0.1 μA. FIG. 7 shows the relationship betweenthe LDH activity and the current increase amount. Thus, the linearrelationship is recognized between the current values in the range of0.1 to 2 U/ml in LDH activity. It is found that it is possible tomeasure the LDH activity by the coenzyme immobilized electrode.

A case where the coenzyme-enzyme immobilized electrode is used as theelectrode 7 to measure the substrate concentration will be describedhereinafter. The electrode 7 is set to a constant-potential of 0.4 Vwith respect to the electrode 8, using a potentionstat. Add the lacticacid into the buffer solution and the anodic current flowing between theelectrode 7 and the counter electrode 9 increases.

FIG. 8 shows the anodic current response when the lactic acid has beenadded so that the lactic acid concentration in the buffer solution maybecome 10⁻³ mol per l. The current shows a steady state value inapproximately one minute and the current increase of 10 μA isrecognized.

FIG. 9 shows the relationship between the lactic acid concentration andthe current increase amount. Thus, the linear relationship between theconcentration and the current value is recognized in the range of 10⁻⁴to 10⁻³ mol per l in lactic acid concentration. It is found that theestimation of the lactic acid can be made by the electrode used herein.

EMBODIMENT 2

SnO₂ nesa-glass is used as the electron collector and carrier, glutamatedehydrogenase (GDH) as the enzyme and NAD as the coenzyme.

The surfaces of the nesa-glass are chemically modified in the procedureshown in the following equation. ##STR3##

NAD alone is, or NAD and GDH are simultaneously, diazocoupled withrespect to the nesa-glass derivative (II), thus providing an electrodewherein the coenzyme alone or the coenzyme and enzyme are immobilized onthe nesa-glass through covalent bonding. FIG. 10 shows a typical schemeof the coenzyme-enzyme immobilized electrode.

Measurement of the GDH activity of 0.1 to 1.3 U/ml could be made, in thesame manner as in the embodiment 1, by the NAD immobilized electrodethus made. Also, according to the similar measurement as the embodiment1 using the NAD-GDH immobilized electrode, the current increase ofapproximately 15 μA was recognized with respect to the glutamic acidconcentration of 1×10⁻³ mol per l. The current reached the steady statevalue in 0.5 minute after the addition of the glutamic acid. And theestimation of the glutamic acid could be made in the concentration rangeof 1×10⁻⁴ to 3×10⁻³ mol per l.

EMBODIMENT 3

Graphite is used as the electron collector, isocitrate dehydrogenase(ICDH) as the enzyme, and nicotine amide adenine dinucleotide phosphate(NADP) as the coenzyme. The graphite powder surfaces are nitrated andfurthermore are reduced. ##STR4##

On the other hand, the carboxyl group is introduced into the NADP in thesame manner as in the embodiment 1. Furthermore, the NADP derivativereacts with the graphite derivative under the existence of DCC and isimmobilized on the graphite surface by the amide bond. ##STR5##

The NADP immobilized graphite powders are press-molded into plate shapeto make the NADP immobilized electrode. In addition, the electrode isdipped in a solution containing ICDH and thereafter is dried and theglutaraldehyde is added thereby to further immobilize enzyme on thegraphite electron collector.

In the same manner as in the embodiment 1, the ICDH activity of 0.1 to0.6 U/ml could be measured by the NADP immobilized electrode made asdescribed hereinabove. Also, the coenzyme-enzyme immobilized electrodeshowed an increase of current of approximately 2.5 μA with respect tothe concentration increase of isocitric acid of 1×10⁻³ mol per l andreached the steady state current after one minute. And the estimation ofthe isocitric acid could be made in the concentration range of 1×10⁻⁴ to1×10⁻³ mol per l.

EMBODIMENT 4

The NAD was used as the coenzyme, albumin as the carrier and graphite asthe electron collector.

After the NAD and albumin have been mixed with a small quantity ofwater, glutaraldehyde is added to the mixture and the NAD can beimmobilized on the albumin surface. The reaction is considered to occurthrough the cross-linking of the amino group of the NAD and SH groups ofalbumin by the glutaraldehyde.

The NAD immobilized albumin powders made as described hereinabove aremixed with the graphite powders and are press-molded to make thecoenzyme immobilized electrode.

In addition, the NAD immobilized electrode is dipped in a solutioncontaining GDH. After the complex of the NAD and the enzyme has beenformed, the glutaraldehyde is further added to cross-link the enzyme.FIG. 11 shows the construction of the NAD-GDH immobilized electrodewhere the carrier albumin is 6. The same measurement, as in theembodiment 1, by use of the above-described coenzyme immobilizedelectrode, showed that the linear relationship was obtained between thecurrent values in the range of 0.1 to 1.5 U/ml.

Also, the same measurement as in the embodiment 1 by the above-describedNAD-GDH immobilized electrode showed that the current increase ofapproximately 6 μA was obtained with respect to the increase in theconcentration of the glutamic acid of 1×10⁻³ mol per l and the currentreached the steady state value after one minute. And the estimation ofthe glutamic acid in the concentration range of 2×10⁻⁴ to 3×10⁻³ mol perl could be made.

EMBODIMENT 5

Alcohol dehydrogenase (ADH) was used as the enzyme, the NAD as thecoenzyme, silica glass powders of approximately 0.05 μm in graindiameter as the carrier and the graphite as the electron collector.First, silica glass surfaces are chemically modified through theprocedure as shown in the following equation. ##STR6##

The NAD alone is, or NAD and ADH are simultaneously, diazo-coupled withthe silica glass derivative (III), whereby the silica glass immobilizesthe coenzyme alone or the coenzyme and enzyme. The above-described twotypes of glass powders are mixed with the graphite powders and arepress-molded, respectively, whereby the coenzyme and coenzyme-enzymeimmobilized electrode are made. FIG. 12 shows the typical constructionscheme of the electrode by the use of glass powders 6.

The similar measurement as in the embodiment 1 by the use of the NADimmobilized electrode indicated that the linear relationship wasobtained between the current values in the range of 0.5 to 3 U/ml inADH.

Also, according to the similar measurement as in the embodiment 1 by theuse of the NAD-ADH immobilized electrode, the current increase of 5 μAwith respect to ethanol of 1×10⁻³ mol per l was recognized and thecurrent reached the steady state value after 0.5 minute. And theestimation of the ethanol in the concentration range of 1×10⁻⁴ to 2×10⁻³ mol per l could be made.

According to the present invention, the activity of the enzyme can bemeasured by the use of the electrode with the coenzyme alone beingimmobilized, without combination with the immobilized enzyme. Thisoperation is impossible to be made by the conventional system where thecoenzyme is retained by the use of the semipermeable membrane.

Namely, even if the coenzyme is retained in the semipermeable membrane,the enzyme itself is macro-molecular. Thus, the enzyme cannot diffuseinto the membrane and cannot make a complex with the coenzyme, with theresult that the reaction shown in FIG. 1 does not occur. In theconventional electrode, it is indispensable to use the semipermeablemembrane to immobilize the coenzyme. On the contrary, in the presentinvention, the semipermeable membrane is not required for theimmobilization of the coenzyme. Since the enzyme in the solution canform a complex with the coenzyme, the activity of the enzyme can bemeasured.

In addition, according to the coenzyme-enzyme immobilized electrode ofthe present invention, the current value provided is larger and the timerequired to reach the steady state current value is shorter as comparedwith the conventional electrode. This fact is apparent throughcomparison in typical construction view between the conventionalelectrode and the electrode of the present invention.

Namely, according to the conventional electrode shown in FIG. 2, thecoenzyme immobilized on the semipermeable membrane is only brought intocontact against the electron collector physically through approaching ofthe membrane to the electron collector. The probability of contactbetween the coenzyme and the electron collector is considerably small.In the case of FIG. 3, the coenzyme is combined with the macromolecularcarrier, and the probability of the enzyme itself coming into contactagainst the electron collector is small due to the steric hindrancebetween the macromolecular carrier and the electron collector.

In the electrode of the present invention shown in FIGS. 4, 10, 11 and12, the oxidation can be efficiently made, since the covalent bond 4always keeps the coenzyme in electrical contact with the electroncollector, even in the case where the coenzyme is immobilized on acarrier of a material different from the electron collector.

In the conventional embodiment, it is considered that the sufficientresponse current value and response time could not be provided due tonot only the reducing effect in the contact probability between thecoenzyme and the electron collector, but also the resisting effect withrespect to diffusion of the substrate through the semipermeablemembrane.

In the foregoing embodiments of the present invention, conductivematerials such as carbon and SnO₂ were provided as examples of theelectron collector. In addition, metal oxides such as RuO₂, In₂ O₃ andWO₃ can be used as the electron collector in the present invention. NADand NADP can be immobilized on the chemically modified surface of theseelectron collectors. Also, although albumin and glass were provided asexamples of the carrier, high molecular organic compounds such asagarose and cellulose, and inorganic compounds such as alumina, silicaalumina and zeolite, can be used. If there are no functional groups onthe surface of these carriers, they can be used after addition of afunctional group which allows covalent bonding to be achieved throughchemical modification to immobilize the coenzyme.

While the present invention has been disclosed in terms of specificembodiments thereof, it is not intended that it be limited thereto, butrather only to the extent set forth hereinafter in the claims whichfollow.

What is claimed is:
 1. A process for preparing a coenzyme immobilizedelectrode, which comprises (1) providing a coenzyme of oxidoreductase,said coenzyme being selected from the group consisting of NAD and NADP,(2) chemically modifying an electron collector, and (3) immobilizingsaid coenzyme on said electron collector through covalent bonding, so asto perform electron transfer with said electron collector.
 2. A coenzymeimmobilized electrode comprising at least a coenzyme of oxidoreductaseand an electron collector, said coenzyme, which is selected from thegroup consisting of NAD and NADP, being immobilized through covalentbonding on said electron collector, said immobilized coenzyme being inelectrical contact with said electron collector.
 3. A coenzymeimmobilized electrode in accordance with claim 2, wherein said coenzymeis immobilized on a chemically modified surface of said electroncollector.
 4. A coenzyme immobilized electrode in accordance with claim2 or 3, wherein said electron collector is selected from the groupconsisting of carbon and a conductive metal oxide.
 5. A coenzymeimmobilized electrode in accordance with claim 4, wherein saidconductive metal oxide is selected from the group consisting of SnO₂,RuO₂, In₂ O₃ and WO₃.